Additional file 4: of Targeted next-generation sequencing identifies clinically relevant somatic mutations in a large cohort of inflammatory breast cancer

Figure S2. Comparison of biological pathway between IBC and non-IBC in four subgroups. (a) The percentage of samples with alteration on 10 biological pathways in the TNBC subgroup; (b) the percentage of samples with alteration on 10 biological pathways in the HR–/HER2+ subgroup; (c) the percentage of samples with alteration on 10 biological pathways in the HR+/HER2– subgroup; (d) the percentage of samples with alteration on 10 biological pathways in the HR+/HER2+ subgroup. The gray bars indicate non-IBC, the black bars indicate IBC; *p < 0.05, **p < 0.01, ***p < 0.001. (PDF 41 kb

Additional file 3: of Targeted next-generation sequencing identifies clinically relevant somatic mutations in a large cohort of inflammatory breast cancer

Figure S1. Comparison of somatic mutation frequency between IBC and non-IBC in four subgroups. (a) The percentage of samples with somatic mutation in the TNBC subgroup; (b) the percentage of samples with somatic mutation in the HR–/HER2+ subgroup; (c) the percentage of samples with somatic mutation in the HR+/HER2– subgroup; (d) the percentage of samples with somatic mutation in the HR+/HER2+ subgroup. The gray bars indicate non-IBC, the black bars indicate IBC; *p < 0.05, **p < 0.01, ***p < 0.001. (PDF 52 kb

Additional file 10: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S8. Metastasis-free survival analysis from the publicly available NKI datasets of breast tumors. a Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 12), High-ETV4 and Low-MMP13 (n = 243), or High-ETV4 and High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. b Metastasis-free survival (MFS) curves for breast tumor patients according to Low-MMP13 (n = 255) or High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. c Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 13) and High-ETV4 (n = 251) mRNA levels. ****P ≤ 0.0001. (PDF 19 kb

Additional file 6: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S4. Expression of MMP13 in MMT cells overexpressing or repressing MMP13. a and b Relative MMP13 mRNA expression in the MMT-Ctrl and MMT-MMP13 (a) or MMT-shCtrl and MMT-shMMP13 cells (b) determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-Ctrl cells was arbitrarily = 1. Error bars indicate SD. ****P ≤ 0.0001. c Western blot analysis of MMP13 protein expression (60 kDa) in the MMT-Ctrl and MMT-MMP13 cells. GAPDH expression served as the loading control. d Zymographic analysis of MMP13 protein activity (55 kDa) from the supernatant of MMT-Ctrl and MMT-MMP13 cells. (PDF 72 kb

Additional file 7: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S5. Expression of ETV4 in MMT-shMMP13-repressing cells. a Relative ETV4 mRNA expression in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-Ctrl + shCtrl cells was arbitrarily = 1. Error bars indicate SD. The results were not statistically significant. b Western blot analysis of ETV4 protein expression (61 kDa) in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells. GAPDH expression served as the loading control. (PDF 71 kb

Additional file 8: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S6. The repression of MMP13 reduces the anchorage-independent growth capacity of MMT-ETV4-overexpressing cells. a Relative MMP13 mRNA expression in the transiently transfected MMT-siCtrl and MMT-siMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-siCtrl cells was arbitrarily = 1. Error bars indicate SD. ****P ≤ 0.0001. b Anchorage-independent growth. MMT-ETV4-siCtrl and MMT-ETV4-siMMP13 cells were cultured for 10 days in soft agar. This histogram represents the number of clones counted for experimental time points. Soft agar assays were conducted three times in triplicate. Magnification × 5. Error bars indicate SD. ****P ≤ 0.0001. (PDF 45 kb

TNF-α up-regulates the expression of the same 20 NF-κB associated genes in HMEC and HUVEC.

<p>Cells were incubated in the presence of TNF-α (40 ng/ml) for 4 h. Gene expression was measured by RT-PCR, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021589#s2" target="_blank"><i>Materials and Methods</i></a>. For each gene, mRNA levels were normalized such that the value for the “basal mRNA level” (smallest amount of target gene mRNA quantifiable, Ct = 35) was 1. Ratio of the mRNA content in stimulated cells <i>versus</i> unstimulated cell mRNA content was calculated. Results are expressed as the mean of two independent experiments.</p

Kinetics of selected NFkB gene expression in TNF-α treated HMEC.

a<p>Ratio of the mRNA content in cells stimulated with TNF-α (40 ng/ml) for 2, 8, 15, 30 min or 1, 4, 24 and 72 h to the mRNA content in the control cell.</p

List of the 55 selected genes tested.

<p>List of the 55 selected genes tested.</p

Inhibitory effects of Bay 11–7082 and IKK2 inhibitor V on TNF-α-induced genes in HMEC endothelial cells.

<p>Cells were pre-treated with pharmacological inhibitors of NF-κB, Bay 11–7082 10 µM or IKK2 inhibitor V (1 and 10 µM), for 30 min before incubation with 40 ng/ml TNF-α for 4 h. mRNA levels were normalized to unstimulated cells which values were set to 1.</p